Gene therapy and nucleic acid immunisation are promising approaches for the treatment and prevention of both acquired and inherited diseases. These techniques provide for the transfer of a desired nucleic acid into a subject with subsequent in vivo expression. Transfer can be accomplished by transfecting the subject's cells or tissues ex vivo and reintroducing the transformed material into the host. Alternatively, the nucleic acid can be administered in vivo directly to the recipient. However, the in vivo delivery method must allow the nucleic acid to enter the cells of the recipient so that the nucleic acid can be expressed.
A number of methods have been developed for gene delivery in these contexts. Of these, transdermal delivery of nucleic acids provides many advantages over oral or parenteral delivery techniques. In particular, transdermal delivery provides a safe, convenient and noninvasive alternative to traditional administration systems, conveniently avoiding the major problems associated with oral delivery (e.g. variable rates of absorption, gastric degradation and metabolism, hepatic first pass effect, gastrointestinal irritation and/or bitter or unpleasant drug tastes) or parenteral delivery (e.g. needle pain, the risk of introducing infection to treated individuals, the risk of contamination or infection of health care workers caused by accidental needle-sticks and the disposal of used needles).
However, transdermal delivery of nucleic acids also presents a number of inherent problems. Passive delivery through intact skin entails the transport of molecules through a number of structurally different tissues. These may include the stratum corneum (the major barrier), the viable epidermis, the papillary dermis or the capillary walls in order to gain entry into the blood or lymph system. Transdermal delivery systems must therefore be able to overcome the various resistances presented by each type of tissue.
Therefore, a number of alternatives to passive transdermal delivery have been developed. These alternatives include the use of skin penetration enhancing agents or “permeation enhancers” to increase skin permeability, as well as non-chemical modes such as the use of ionophoresis, electroporation or ultrasound. However, these alternative techniques often give rise to their own side effects such as skin irritation or sensitization.
Recently, particle-mediated techniques suitable for transdermal delivery of nucleic acids have been developed. Particles bearing the nucleic acid of interest are accelerated to high velocity and fired into target tissue using a particle accelerating device. In vivo, the particles may be fired directly into recipient cells, avoiding the need for cell uptake of the passenger nucleic acid.
Various particle acceleration devices suitable for particle-mediated delivery are known in the art. Existing devices employ an explosive, electric or gaseous discharge to propel the coated carrier particles towards target cells. The Biolistic® device, for example, delivers DNA-coated microscopic gold beads directly into the cells of the epidermis (Yang et al (1990) PNAS USA 87:9568-9572). Particles can also be delivered using a needleless syringe device such as that described in U.S. Pat. No. 5,630,796 to Bellhouse et al (“the PowderJect® needleless syringe device”).
Particle-mediated devices are intended to allow safe and easy delivery of nucleic acids. However, the physical characteristics of the particles need to be engineered to meet the demands of needleless administration, in which particles are typically fired at very high velocities. The particles need to have a structural integrity such that they can survive the action of, for example, a gas jet of a syringe or ballistic impact with skin or mucosal tissue. It is also important that the particles have a density that enables the particles to achieve sufficient momentum to penetrate tissue. For nucleic acid delivery however, the particles should be smaller than cell size so that they can penetrate cell membranes without disrupting the cells. Nucleic acids are themselves susceptible to degradation on storage. Therefore, the nucleic acid needs to be maintained in stable conditions when associated with the particle. However, the association of the nucleic acid with the particle should also allow efficient expression of the nucleic acid after delivery to the target cell. Where the nucleic acid encodes an antigen, the means of particle association should also allow immunogenicity of the antigen in a subject.
According to one technique, particles suitable for particle mediated delivery can be formed by coating nucleic acid molecules onto inert metal carrier particles. The carrier particles are selected from materials having a suitable density and size, such as tungsten or gold. A number of methods are known for coating or precipitating DNA or RNA onto gold or tungsten particles. These methods generally combine a predetermined amount of gold or tungsten with plasmid DNA, CaCl2 and spermidine. The resulting solution is vortexed continually during the coating procedure to ensure uniformity of the reaction mixture. After precipitation of the nucleic acid, the coated particles can be transferred to suitable membranes and allowed to dry prior to use, coated onto surfaces of a sample module or cassette, or loaded into a delivery cassette for use in particular particle-mediated delivery instruments.